predicting the important enzyme players in human breast ...€¦ · 26 whether these enzymes are...

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Article

Predicting the important enzyme players in human breast milk digestionNora Khaldi, Vaishnavi Vijayakumar, David C. Dallas, Andrés Guerrero, Saumya Wickramasinghe,Jennifer T. Smilowitz, Juan F. Medrano, Carlito B. Lebrilla, Denis C Shields, and J. Bruce German

J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf405601e • Publication Date (Web): 12 Mar 2014

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1

Title. Predicting the important enzyme players in human breast milk digestion 1

2

Nora Khaldia,b,c,1

, Vaishnavi Vijayakumara,b

, David C. Dallasc,d

, Andrés Guerreroe, 3

Saumya Wickramasinghef,g

, Jennifer T. Smilowitzc,d

, Juan F. Medranog, Carlito B. 4

Lebrillae, Denis C. Shields

a,b, and J. Bruce German

c,d 5

6

aConway Institute of Biomolecular and Biomedical Research, School of Medicine and 7

Medical Sciences. University College Dublin, Dublin, Republic of Ireland 8

bComplex and Adaptive Systems Laboratory, University College Dublin, Dublin, 9

Republic of Ireland 10

cDepartment of Food Science and Technology, University of California, Davis, 11

California 95616, USA. 12

dFoods for Health Institute, University of California, Davis, California 95616, USA. 13

eDepartment of Chemistry University of California, Davis Califorina 95616, USA. 14

fDepartment of Basic Veterinary Sciences, University of Peradeniya, Sri Lanka 15

gDepartment of Animal Science, University of California, Davis, California 95616, 16

USA. 17

18

1To whom correspondence should be addressed: Nora Khaldi, [email protected], 19

phone: 00353 -1-7165335, UCD Complex and Adaptive Systems Laboratory, 20

University College Dublin, Dublin 4, Ireland. 21

22

23

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Journal of Agricultural and Food Chemistry

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ABSTRACT 24

Human milk is known to contain several proteases, but little is known about 25

whether these enzymes are active, which proteins they cleave and their relative 26

contribution to milk protein digestion in vivo. We analyzed the mass spectrometry-27

identified protein fragments found in pooled human milk by comparing their cleavage 28

sites with the enzyme specificity patterns of an array of enzymes. The results indicate 29

that several enzymes are actively taking part in the digestion of human milk proteins 30

within the mammary gland, including plasmin and/or trypsin, elastase, cathepsin D, 31

pepsin, chymotrypsin, a glutamyl endopeptidase-like enzyme and proline 32

endopeptidase. Two proteins were most affected by enzyme hydrolysis: β-casein and 33

polymeric immunoglobulin receptor. In contrast, other highly abundant milk proteins 34

such as α-lactalbumin and lactoferrin appear to have undergone no proteolytic 35

cleavage. We also show that a peptide sequence containing a known anti-microbial 36

peptide is released in breast milk by elastase and cathepsin D. 37

38

39

KEYWORDS: hydrolysate, human milk digestion, milk, nutrition, proteolytic 40

enzymes, bioactive peptide. 41

42

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INTRODUCTION 43

Milk is a live secretion that contains numerous complex biomolecules such as 44

proteins, oligosaccharides and lipids. In addition to these components, previous 45

studies have shown that milk also contains active and inactive forms of hydrolytic 46

enzymes capable of acting upon these biopolymers, including proteolytic enzymes. 47

These independent studies have shown that milk contains plasmin (1, 2); 48

immunoreactive anionic trypsin, most likely present in complex with IgA (3, 4); and 49

cathepsin D (5). 50

Although it has been established that proteolytic enzymes are present in milk, 51

little is known about whether these enzymes are active on milk proteins. In addition, 52

if the enzymes were to be active, their contribution to milk protein hydrolysis, and 53

their relative contribution compared to one another are unknown. 54

Many of the studies of milk proteolytic enzymes were carried out on bovine 55

milk, mainly for cheese- or mastitis-related questions; very few were carried out on 56

human milk (6-9). Most milk proteolytic enzyme investigations have been carried out 57

separately for each enzyme and do not determine the specific effects of each enzyme 58

on milk proteins. This lack of research means that the impact the enzymes have on 59

human milk proteins remains largely unknown. The biological value of these 60

proteolytic enzymes in milk remains ambiguous. Some reports suggest that milk 61

enzymes may support infant growth and nutrition (10). Some studies suggest that 62

these enzymes may release bioactive peptides that are beneficial for the infant’s 63

development (see reviews (11, 12)). But, so far, none of these peptide actions and in 64

vivo catalytic releases have been demonstrated. 65

Understanding milk composition, and its diverse catalytic activities, notably 66

the proteolytic enzymes, will shed light on mammalian development, evolution, and 67

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how to protect neonates. A new generation of analytical and computational tools has 68

made it possible to investigate milk’s biopolymers in greater breadth and detail. We 69

previously published a novel mass spectrometry (MS)-based peptide search platform 70

that we used in an iterative searching strategy to identify peptides in minor 71

abundances in human milk (13). We discovered many peptides, proving that milk 72

proteins can be digested prior entering the infant’s digestive system. Many of these 73

peptides overlapped with known antimicrobial peptides (13). In this study, we assess 74

the proteolytic activity in human breast milk. We used computational tools to analyze 75

the peptide fragments given their milk protein sequence context. Using these tools, we 76

predicted the proteolytic enzymes that are active in human milk, we examined the 77

relative contribution of each enzyme, and we identified the proteins that have been the 78

most cleaved by the predicted enzymes. mRNA expression was also measured in 79

human milk to determine whether these enzymes are expressed in the mammary gland 80

or whether they enter milk from other sources such as blood. 81

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MATERIALS AND METHODS 82

Breast milk collection: Mature breast milk was collected from two healthy mothers 83

who delivered at term (IRB number 216198). The milk was kept in storage up to 1 84

month. There is no impact on milk proteins and enzymes with storage. In other words, 85

no enzyme activity occurs when samples are frozen. The stage of lactation of the first 86

mother was 3 months and the second was 4 months. The mothers providing this milk 87

were instructed to cleanse each breast with water and then use an electronic pump to 88

collect a pool of milk from both breasts. The samples were then stored in the subjects' 89

freezers and delivered to the laboratory on ice where they were stored at -80C. 90

91

Sample preparation: Peptides were extracted from human milk according to the 92

method of Dallas et al. (13). Briefly, 200 µL of each milk sample were thawed on ice 93

and combined. To remove milk fat globules, the milk was centrifuged at 3,000 x g for 94

10 min and the skim infranate was extracted. Centrifugation was repeated on the skim 95

infranate to remove any remaining visible lipid layer. Proteins were then precipitated 96

with addition of 400 µL of 200 g/L trichloroacetic acid. Samples were vortexed 97

briefly, then centrifuged at 3,000 x g for 10 min and the peptide-containing 98

supernatant was collected, leaving the precipitated protein. This precipitation was 99

repeated for a total of three times. Trichloroacetic acid, salts, oligosaccharides and 100

lactose were then removed from the peptides by C18 reverse-phase preparative 101

chromatography. Contaminants were eluted with water and peptides were then eluted 102

with 80% acetonitrile (ACN), 0.1 % trifluoroacetic acid (v/v). The peptide solution 103

was then dried down in a vacuum centrifuge at 37ºC. After drying, the sample was 104

rehydrated in 40 µL of nanopure water for MS analysis. 105

106

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MS analysis of human breast milk peptides: Peptides were analyzed via nano-107

liquid chromatography chip quadrupole time-of-flight tandem MS (Agilent, Santa 108

Clara, CA). Two µL of sample were injected for each run onto the C18 reverse-phase 109

nano chip. The nanopump flow was .3 µL/min and the capillary pump flow rate was 3 110

µL/min. Peptides were eluted with the following gradient of solvent A (3% ACN, 111

0.1% formic acid (FA) (v/v)) and solvent B (90% ACN, 0.1% FA (v/v)): 0–8% B 112

from 0–5 min, 8–26.5% B from 5–24 min, 26.5–100% B from 24–48 min, followed 113

by 100% B for 2 min and 100% A for 10 min (to re-equilibrate the column). The 114

instrument was run in positive ionization mode. Data collection thresholds were set at 115

200 ion counts or 0.01% relative intensity for MS spectra and 5 ion counts or 0.01% 116

relative intensity for MS/MS. Data were collected in centroid mode. The drying gas 117

was 350 °C and flow rate was 3 L/min. The required chip voltage for consistent spray 118

varied from 1850 to 1920 V. Automated precursor selection based on abundance was 119

employed to select peaks for tandem fragmentation with an exclusion list consisting 120

of all peptides identified in previous analyses in this study. The acquisition rate 121

employed was 3 spectra/s for both MS and MS/MS modes. The isolation width for 122

tandem analysis was 1.3 m/z. The collision energy was set by the formula 123

(Slope)*(m/z)/100+Offset, with slope = 3.6 and offset = -4.8. Five tandem spectra 124

were collected after each MS spectrum, with active exclusion after 5 MS/MS for 0.15 125

min. Precursor ions were only selected if they had at least 1000 ion counts or 0.01% 126

of the relative intensity of the spectra. Mass calibration was performed during data 127

acquisition based on an infused calibrant ion with a mass of 922.009789 Da. 128

Agilent Mass Hunter Qualitative Analysis Software (Santa Clara, CA) was used to 129

analyze the data obtained. Molecules identified in the spectral analysis were grouped 130

into compounds by the Find by Molecular Feature algorithm, which groups together 131

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molecules across charge state and charge carrier. All tandem-MS from each data file 132

were exported as Mascot Generic Files (.mgf) with a peptide isotope model and a 133

maximum charge state of +9. 134

Peptide identification was accomplished using both the MS-GFDB (via a command-135

line interface) and X!Tandem (using the downloadable graphical user interface). The 136

human milk library used in both searches was constructed based on a query to the 137

Uniprot database. The query returned only proteins from Homo sapiens and at least 138

one of the following: “tissue specificity” keyword “milk” or “mammary”, “tissue” 139

keyword “milk” or “mammary” or gene ontology “lactation”. This query returned a 140

list of 1,472 proteins. These were exported to FASTA file format. For MS-GFDB, 141

peptides were accepted if p-values were less than or equal to 0.01 corresponding to 142

confidence levels of 99%. No p-values exist in X!Tandem, so a closely related 143

statistic, e-value, was used for the X!Tandem search. The e-value thresholds selected 144

were again 0.01, reflecting 99% confidence. In both programs, masses were allowed 145

20 ppm error. No complete (required) modifications were included but up to four 146

potential modifications were allowed on each peptide. Potential modifications 147

allowed were phosphorylation of serine, threonine or tyrosine and oxidation of 148

methionine. A non-specific cleavage ([X]|[X]) (where ‘X’ is any amino acid) was 149

used to search against the protein sequences. For MS-GFDB, the fragmentation 150

method selected in the search was collision-induced dissociation and the instrument 151

selected was time-of-flight. For X!Tandem, there was no option for fragmentation 152

type and instrument selection. Because the instrument did not always select the 153

monoisotopic ion for tandem fragmentation, isotope errors were allowed (allowing up 154

to one C13). No model refinement was employed in X!Tandem. 155

156

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Enzyme prediction: The web-based software EnzymePredictor (14) was employed to 157

evaluate and predict which enzymes most likely contributed to cleavage of human 158

breast milk proteins (http://bioware.ucd.ie/~enzpred/Enzpred.php). Enzymes were 159

classified based on their total number of performed cleavages, and they were 160

evaluated based on their odds ratio (OR; See Table 1), which is an indicator of their 161

degree of participation in the hydrolysis or the proteins. The OR values indicate that 162

certain enzymes are over-represented, and others under-represented. The following 163

information were also collected from EnzymePredictor: number of times an enzyme 164

could have cleaved within the current peptides, total number of proteins cleaved by 165

each enzyme, total number of cleavages performed by an enzyme on the C- and N-166

terminus. 167

168

Expression profiling of human milk 169

Fresh milk sample collection: Fresh milk samples were obtained from three healthy 170

females on days 4, 15, 30 and 60 postpartum who gave birth to a term infant (> 37 171

weeks of gestation). In the early morning period, the donor manually pumped one 172

breast until emptied into a collection bag, and immediately delivered on cold-packs to 173

the lab for processing. The Institutional Review Board of University of California, 174

Davis, approved the project. 175

176

177

RNA extraction for gene expression studies: Somatic cells were pelleted by adding 178

50 µL of 0.5 M ethylenediaminetetraacetic acid to 20 mL of fresh milk and 179

centrifuged at 2,000 x g at 4˚C for 10 min (15). The pellet of cells was washed with 180

10 mL of phosphate-buffered saline at pH 7.2 and 10 µL of 0.5 M 181

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ethylenediaminetetraacetic acid (final concentration 0.5 mM) and filtered through 182

sterile cheesecloth to remove any debris. The cells were then centrifuged again at 183

2,000 x g at 4˚C for 10 min. The supernatant was decanted and RNA was extracted 184

from the milk somatic cell pellet using Trizol (Invitrogen, Carlsbad, CA) according to 185

the manufacturer’s instructions. RNA was quantified by an ND-1000 186

spectrophotometer (Fisher Thermo, Wilmington, MA) and the quality and integrity 187

was assessed by the spectrophotometer 260/280 ratio, gel electrophoresis and by 188

capillary electrophoresis with an Experion Bio-analyzer (Bio-Rad, Hercules, CA). 189

190

RNA sequencing and data analysis: Gene expression analysis was conducted on 191

fresh milk samples collected on days 4, 15, 30 and 60 postpartum by RNA sequencing 192

(RNA-Seq). Messenger RNA was isolated and purified using an RNA-Seq sample 193

preparation kit (Illumina, San Diego, CA). The fragments were purified and 194

sequenced at the UC Davis Genome Center DNA Technologies Core Facility using 195

the Illumina Genome Analyzer (GAII) and Illumina HiSeq 2000. Sequence reads 196

were assembled and analyzed in RNA-Seq. Expression analysis was performed with 197

the CLC Genomics Workbench 6.0 (CLC Bio, Aarhus, Denmark). Human Genome, 198

GRCh37.69 (ftp://ftp.ensembl.org/pub/release-69/genbank/homo_sapiens/) was 199

utilized as the reference genome for the assembly. Data were normalized by 200

calculating the ‘reads per kilobase per million mapped reads’ (RPKM) (16) for each 201

gene and annotated with ENSEMBL human genome assembly (55,203 unique genes). 202

203

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RESULTS AND DISCUSSION 204

This project aimed to determine the enzymes responsible for the cleavage 205

patterns identified in milk; examine each enzyme’s contribution to the hydrolyses of 206

human milk proteins; and clarify whether these enzymes are expressed in milk or 207

enter milk from other sources. 208

A novel MS-based peptide search platform using an iterative searching 209

strategy detected a variety of peptides in minor abundances in pooled human breast 210

milk samples (13). The peptides extracted and identified for the present study were 211

analyzed using EnzymePredictor (14). The results of this analysis are presented in 212

Table 1. The enzymes are ordered based on their total number of cleavages, from 213

those that have cleaved extensively to those that are predicted to have cleaved one 214

residue. As discussed in the interpretation of EnzymePredictor (14), the enzymes with 215

a combined high number of total cleavages performed and a high odds ratio are the 216

enzymes that are most likely active in the milk. 217

Expression profiles from samples of human breast milk were established as 218

the means to investigate if the predicted enzymes originate from the mammary 219

epithelial cells or they enter milk from other sources, such as blood. The samples that 220

were obtained for analysis for peptide detection via MS were pooled from mature 221

milk samples from two mothers, both 3-months postpartum, while that of expression 222

data was of 2-month postpartum milk. 223

1. Major cleavage of proteins in human milk is at trypsin and plasmin target 224

sites. 225

The natural cleavages of milk proteins showed a strong enrichment for cleavage after 226

K or R, both consistent with plasmin and trypsin cleavage (Table 1,4). 227

The presence of plasmin—an enzyme that plays a major role in the proteolytic 228

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breakdown of blood clots—has been previously reported in milk (1), but its 229

contribution to human milk proteolytic hydrolysis is unknown. This study found that 230

plasmin potentially cleaved 320 peptides (Fig. 1, Table 1) derived from 15 milk 231

proteins (Table 2). Anionic trypsin's presence in human milk was initially identified 232

by Monti et al. and Borulf et al. (3, 4). Borulf et al. showed that trypsin’s presence in 233

milk is likely in complex with IgA. Whether this enzyme contributes to the hydrolysis 234

of human milk proteins remains unknown. 235

The two proteins most affected by potential plasmin or trypsin digestion were 236

polymeric immunoglobulin receptor (32 cleavages of peptides) and β-casein (89). 237

Other hydrolyzed proteins included αS1-casein (11), osteopontin (18), butyrophilin 238

subfamily 1 member A1 (15), and κ-casein (7). These analyses found that plasmin 239

cleaves an androgen receptor that trypsin failed to cleave (Q9UN21; Table 2-3). 240

Butyrophilin-subfamily 1 member A1 is part of the milk fat globule membrane. Thus, 241

the low number of peptides detected from this protein might be due to the 242

centrifugation for the removal of the milk fat globules. Trypsin can typically not 243

cleave if the K or R is followed by a P, which makes the androgen receptor better fit 244

the plasmin pattern (Table 4). This result would suggest that the cleavage tends to 245

follow the specificity of plasmin more consistently than that of trypsin. However, 246

there are too few cleavages representing K or R followed by P (only one in our case) 247

to disambiguate between plasmin and trypsin activities in the milk. 248

2. Enzymes cleaving hydrophobic target sites: elastase, cathepsin D, pepsin and 249

chymotrypsin. 250

While the active form of cathepsin D is found in bovine milk (5), the major form of 251

this enzyme in milk is the inactive zymogen, procathepsin D (17). In this study, 252

cathepsin D was found to be active in human milk (Table 1), cleaving 130 times. 253

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Cathepsin D hydrolyzed the highest number of milk proteins (24 milk proteins) 254

compared to the other enzymes present in milk (Table 1,5; Figure 1). Although the 255

total number of times cathepsin D performed a cleavage is high (130 total cleavages), 256

most of these are located in two proteins: β-casein with 78 cleavages and polymeric 257

immunoglobulin receptor with 21 cleavages. Interestingly, both these proteins have 258

also been the most digested ones with all 5 top predicted enzymes. 259

A high number of potential cleavage sites was predicted for cathepsin D (846; 260

Table 1), which were not observed in the peptide results, with an Odds Ratio below 261

one. This failure to produce predicted peptides could be due to the positioning of a 262

sub-population of these residues in regions difficult for the enzyme to access due to 263

structural constraints. This structural inhibition is more likely for cathepsin D sites, 264

since many potential cleavage sites are strongly hydrophobic, and hydrophobic 265

regions are usually buried within the structured regions of proteins. 266

Elastase is another enzyme predicted to be active in human milk. Elastase is 267

known to be a major proteinase in polymorphonuclear neutrophils (PMNs), which are 268

phagocytic cells that destroy infectious agents in humans (18). Elastase activity has 269

been detected in PMNs recovered from milk during experimentally induced mastitis 270

(19). There is substantial overlap between the cleavage sites of elastase and cathepsin 271

D, and again there are a large number of sites (563) interior to the peptides that were 272

not cleaved. Elastase potentially performed 90 total cleavages of milk peptides (Table 273

6) over 13 proteins. The proteins that elastase cleaved the most are β-casein (41 274

cleavages of unique peptides) and polymeric immunoglobulin receptor (25 cleavages 275

of unique peptides). No traces of elastase activity were observed on κ-casein (Table 276

6), and only one cleavage for cathepsin D (Table 5). This reflects the similar, but not 277

identical, cleavage pattern between elastase and cathepsin D. To resolve the 278

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specificity, the number of sites that were cleaved in common between both were 279

determined (60 cleavages of unique peptides) or specific to only one (69 for cathepsin 280

D of unique peptides, and 28 for elastase of unique peptides). This pattern suggests a 281

role for both enzymes in human milk digestion prior to infant consumption. 282

Two other enzymes with hydrophobic targets, pepsin and chymotrypsin, are 283

consistent with a number of digestion sites additional to those digestible by cathepsin 284

D and elastase. Only 21 of the 75 total peptide terminal cleavage sites of unique 285

peptides that are predicted to be performed by chymotrypsin are common cleavages 286

with elastase and cathepsin D. For pepsin, only 38 of the 72 cleavages sites predicted 287

from the unique peptides are shared with elastase and cathepsin D. Furthermore, only 288

16 peptide terminal cleavage sites are shared between both pepsin and chymotryspin. 289

These findings indicate the first support that pepsin- and chymotrypsin-like activities 290

are present in human breast milk (Table 1). 291

3. Examination of gene and protein expression profile of proteases in human 292

milk. 293

Previous work has suggested that plasmin is found in milk but that it 294

originates from blood (20). This study tested if the enzymes responsible for the 295

observed peptide fragments are produced in the mammary gland or migrate into milk 296

from other origins. Gene expression analysis was carried out for human milk 297

according to the procedure used for bovine milk ((15); Table 1). The expression was 298

analyzed for days 4, 15, 30 and 60 of lactation. For consistency purposes, day 60 gene 299

expression was determined, however we also took into account the expression levels 300

for the other days of lactation to build Table 1 which shows the possibility of 301

expression of an enzyme at different stages of lactation. The results show that very 302

few of the proteolytic enzymes described in Table 1 are expressed in the mammary 303

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gland at these timepoints (Table 1). In agreement with previous literature, no gene 304

expression of plasmin was detected in the milks. Moreover, no expression was 305

observed for the intestinal enzyme trypsin. 306

Cathepsin D was highly expressed (365 RPKM, Table 1). Elastase, however, 307

was not expressed in the mammary epithelial cells (Table 1). As mentioned above, the 308

presence of active elastase in milk may be explained by its presence in PMNs. We 309

would expect the vast majority of PMN cells to be pelleted by the centrifugation and 310

thus, would not participate in any degradation of the milk proteins after centrifugation 311

(see methods). However, PMN can secrete elastase while still within the mammary 312

gland, and the predicted elastase activity may derive from them. 313

4. Other predicted proteases that were also expressed in human milk 314

Most of the other predicted enzymes have a low number of total cleavages, 315

making it difficult to fully support their presence in milk (Table 1; total 316

cleavages<=44). Interestingly, some of these enzymes activities predicted in milk 317

seem to be expressed in matching proteases. Based on a relatively specific cleavage 318

site not shared by other proteases in these analyses (cleaves after E; with 76 cleavages 319

in total), we predicted a glutamyl endopeptidase-like activity. Transcripts for glutamyl 320

endopeptidase were, however, not detected in mammary epithelial cells. A glutamyl 321

endopeptidase-like protease (proteasome subunit beta type-3, PSMB3) is, however, 322

highly expressed (75.9 RPKM; Table 1). This result may explain the high number of 323

cleavage sites predicted to be due to a glutamyl endopeptidase-like enzyme (930). 324

Indeed, this glutamyl endopeptidase-like protease may have a more specific cleavage 325

pattern explaining the many residues containing glutamic acid (E) that it did not 326

cleave, as this cannot be accounted for by a structural bias, since the charged glutamic 327

acids are not found buried in the core of the proteins. While the gene expression of a 328

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glutamyl endopeptidase (called “a disintegrin and metalloproteinase with 329

thrombospondin motifs 4” (ADAMTS4) was detected, its potential cleavage site, 330

E'[AS], is only cleaved eight times in the dataset. Thus, there is no indication for this 331

enzyme playing a role as the major glutamyl endopeptidase activity in milk. 332

Proline-endopeptidase was found to be expressed in milk (5.52 RPKM; Table 333

1). Proline-endopeptidase is responsible for the cleavage of 45 residues covering a 334

total of 18 milk proteins (Table 1; Figure 1). 335

5. In vivo release of antimicrobial peptides in human milk 336

Milk proteins may carry encrypted functional peptide sequences that, when 337

released by enzymes from the intact protein, help in the protection and development 338

of the neonate (12). But without in vivo results, these concepts remain unproven. This 339

study identified four peptides that overlap (2 extra or less amino acids on the N-340

terminus of the peptide) with a known antimicrobial peptide that has been reported 341

previously in the literature (21). This peptide is present at the C-terminus of β-casein 342

(Figure 2). These four overlapping peptides are naturally released in human milk via 343

the action of several enzymes, including cathepsin D and elastase (Figure 2). The 344

overlapping peptides most likely carry the antibacterial activity, as the amino acid 345

additions compared to the literature-defined sequence is unlikely to abolish the 346

antimicrobial activity of these sequences. 347

6. Uneven distribution of enzyme activity in human milk 348

To measure the enzyme activity we considered the unique cleavages of each 349

(i.e. if an enzyme cleaves out the same peptide from the parent protein multiple times, 350

we will only consider this to be one unique cleavage). This measure highlights more 351

the range of cleavages of an enzyme and makes it possible to compare it with other 352

enzyme ranges rather than comparing its ability to cleave many times the same 353

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peptides. Consequently, this measure does not take the abundance of the proteins into 354

account. Using this approach we find that two proteins—β-casein and polymeric 355

immunoglobulin receptor—show the highest susceptibility to the milk enzyme 356

activity, as shown by the large number of fragments found from these two proteins. β-357

casein is a milk-specific protein expressed during lactation. Interestingly, the other 358

two milk-specific proteins—αS1-casein and κ-casein—show little digestion 359

susceptibly within the mammary gland. The possibility of protein structural disorder 360

being the source of variability in degradation susceptibility among these proteins is 361

not supported by these results, as κ-casein and αS1-casein are also highly disordered 362

proteins. The K and R residues are those that show the most susceptibility for the 363

cleavages observed in human milk proteins. However, no enrichment of these amino 364

acids was found in β-casein versus αS1-casein or κ-casein (14 K and R sites in β-365

casein; 16 in αS1-casein; and 13 κ-casein). In fact, there are even less K and R in β-366

casein over the sequence length compared to αS1-casein or κ-casein, arguing that this 367

is not the driver behind β-casein susceptibility, nor is it for polymeric 368

immunoglobulin receptor (6% in β-casein; 9% in α-S1-casein; 8% κ-casein; and 10% 369

in polymeric immunoglobulin receptor). On the other hand, post-translational 370

modifications may also explain these variations in susceptibility of proteins to 371

degradation. Indeed, large modifications on proteins may prevent enzymes from 372

reaching their target site. However, investigating the potential modifications sites 373

using Uniprot shows that all the casein proteins are slightly/equivalently modified (in 374

terms of numbers of glycosylations and phosphorylations). 375

The other major milk-specific proteins that have not been affected are α-376

lactalbumin, and lactoferrin. No peptides from these proteins were detected despite 377

the fact that plasmin, trypsin, cathepsin D and elastase were all predicted to cleave 378

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both of these proteins. It is perhaps because of the particular structure and post-379

translational modifications of α-lactalbumin, and lactoferrin that prevented the 380

enzymes from cleaving them. 381

Although cathepsin D is highly expressed in milk (Table 1), it does not show a 382

high effectiveness in cleaving milk proteins. This lower cleavage rate may be because 383

cathepsin D is most effective at a more acidic pH (pH=5 for cathepsin D), which is 384

different to the more neutral milk pH. 385

Independent studies have shown the existence and activity of some enzymes in 386

milk, but the extent to which these enzymes are active relative to each other remained 387

unknown. Likewise, whether or not these enzymes are created in the mammary 388

epithelial cells or not was unclear. The enzymes that are active in human milk and 389

responsible for protein digestion prior to entry into the infant’s digestive system were 390

determined. Using a combination of bioinformatics, peptidomics, transcriptomics and 391

literature results, we showed that milk begins to be digested even before infant 392

consumption, and that this digestion is mainly carried out by four proteolytic 393

enzymes: plasmin, a trypsin-like enzyme, elastase, and cathepsin D (Tables 1, 2, 3, 5, 394

6). Among these four enzymes, only cathepsin D and elastase are expressed in milk, 395

with elastase showing only weak expression at day 15 and 30, and no expression after 396

that (Table 1). Activity and expression of chymotrypsin and pepsin-like activity were 397

also seen (Table 1). This finding is surprising, given that these enzymes have never 398

been reported in human breast milk. Other enzymes that this analysis predicted from 399

activity and expression are glutamyl endopeptidase and proline endopeptidase (Table 400

1). 401

Interestingly, plasmin, trypsin, elastase and cathepsin D cleave the casein 402

proteins (αs1-, β-, and κ-casein) to various extents. Our novel MS-based peptide 403

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18

search platform using an iterative searching strategy to detect peptides in minor 404

abundances did not detect peptides resulting from α-lactalbumin. Plasmin, a trypsin-405

like enzyme, elastase and cathepsin are predicted to cleave this protein, yet none of 406

the predicted peptides could be detected. The mechanism causing this protein 407

selectivity of digestion remains unknown. 408

409

We know very little about proteolytic enzyme activity in human milk, this makes the 410

problem of humanizing formulae milk even harder. The work we have carried out 411

here sheds light on the different enzymes we find in human milk and the relative 412

contribution of each of them –in terms of the release of unique peptides- to cleaving 413

milk proteins. The example illustrated in figure 2 of a known anti-microbial activity 414

being released illustrates how enzyme activity in human milk may be playing a much 415

greater role than previously anticipated, and fully clarifying this role is key in 416

understanding the infant needs. 417

418

ABBREVIATIONS 419

MS, mass spectrometry; FA, formic acid; ACN, acetonitrile; OD, odds ratio; PMN, 420

polymorphonuclear neutrophils; RPKM, reads per kilobase per million mapped reads. 421

422

ACKNOWLEDGEMENTS 423

The authors thank Prof. Alan Kelly and Kevin Rue for helpful discussions. 424

This work was funded by the Irish Research Council for Science, Engineering and 425

Technology, co-funded by Marie Curie Actions under FP7; Enterprise Ireland grant to 426

Food Health Ireland; SFI grant 08/IN.1/B1864; University of California Discovery 427

Program (05GEB01NHB), the National Institute of Health (HD059127 and 428

Page 18 of 35



19

HD061923) California Dairy Research Foundation; United States Department of 429

Agriculture, National Institute for Food and Agriculture post-doctoral fellowship; 430

National Science Foundation Graduate Research Fellowship Program; National 431

Institute of Health Training Program in Biomolecular Technology (2-T3-GM08799). 432

433

Notes 434

The authors declare no competing financial interest. 435

436

REFERENCES 437

1. Heegaard, C. W.; Larsen, L. B.; Rasmussen, L. K.; Højberg, K. E.; Petersen, 438

T. E.; Andreasen, P. A., Plasminogen activation system in human milk. J. Pediatr. 439

Gastroenterol. Nutr. 1997, 25, 159-166. 440

2. Nielsen, S. S., Plasmin System and Microbial Proteases in Milk: 441

Characteristics, Roles, and Relationship. J. Agric. Food Chem. 2002, 50, 6628-6634. 442

3. Borulf, S.; Lindberg, T.; Mansson, M., Immunoreactive anionic trypsin and 443

anionic elastase in human-milk. Acta Paediatr. 1987, 76, 11-15. 444

4. Monti, J.; Mermoud, A.; Jolles, P., Trypsin in human milk. Experientia 1986, 445

42, 39-41. 446

5. Kaminogawa, S.; Yamauchi, K., Acid protease of bovine milk. Agric. Biol. 447

Chem. 1972, 36, 2351-2356. 448

6. Armaforte, E.; Curran, E.; Huppertz, T.; Ryan, C. A.; Caboni, M. F. O.; 449

Connor, P. M.; Ross, R. P.; Hirtz, C.; Sommerer, N.; Chevalier, F., Proteins and 450

proteolysis in pre-term and term human milk and possible implications for infant 451

formulae. Int. Dairy J. 2010, 20, 715–723. 452

7. Ferranti, P.; Traisci, M. V.; Picariello, G.; Nasi, A.; Boschi, V.; Siervo, M.; 453

Falconi, C.; Chianese, L.; Addeo, F., Casein proteolysis in human milk: tracing the 454

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20

pattern of casein breakdown and the formation of potential bioactive peptides. The 455

Journal of dairy research 2004, 71, 74-87. 456

8. Borulf, S.; Lindberg, T.; Mansson, M., Immunoreactive anionic trypsin and 457

anionic elastase in human milk. Acta paediatrica Scandinavica 1987, 76, 11-5. 458

9. Monti, J. C.; Mermoud, A. F.; Jolles, P., Trypsin in human milk. Experientia 459

1986, 42, 39-41. 460

10. Shahani, K.; Kwan, A.; Friend, B. A., Role and significance of enzymes in 461

human milk. Am. J. Clin. Nutr. 1980, 33, 1861-1868. 462

11. Korhonen, H.; Pihlanto, A., Bioactive peptides: production and functionality. 463

Int. Dairy J. 2006, 16, 945-960. 464

12. Dallas, D. C.; Underwood, M. A.; Zivkovic, A. M.; German, J. B., Digestion 465

of protein in premature and term infants. Journal of Nutritional Disorders and 466

Therapy 2012, 2, 1-9. 467

13. Dallas, D. C.; Guerrero, A.; Khaldi, N.; Castillo, P. A.; Martin, W. F.; 468

Smilowitz, J. T.; Bevins, C. L.; Barile, D.; German, J. B.; Lebrilla, C. B., Extensive in 469

vivo human milk peptidomics reveals specific proteolysis yielding protective 470

antimicrobial peptides. J. Proteome Res. 2013, 12, 2295-2304. 471

14. Vijayakumar, V.; Guerrero, A.; Davey, N.; Lebrilla, C. B.; Shields, D. C.; 472

Khaldi, N., EnzymePredictor: a tool for predicting and visualizing enzymatic 473

cleavages of digested proteins. J. Proteome Res. 2012, 11, 6056-6065. 474

15. Wickramasinghe, S.; Rincon, G.; Islas-Trejo, A.; Medrano, J. F., 475

Transcriptional profiling of bovine milk using RNA sequencing. BMC Genomics 476

2012, 13, 45-58. 477

16. Mortazavi, A.; Williams, B. A.; McCue, K.; Schaeffer, L.; Wold, B., Mapping 478

and quantifying mammalian transcriptomes by RNA-Seq. Nature methods 2008, 5, 479

621-628. 480

17. Larsen, L. B.; Petersen, T. E., Identification of five molecular forms of 481

cathepsin D in bovine milk. In Aspartic Proteinases, Springer: 1995; pp 279-283. 482

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21

18. Travis, J.; Fritz, H., Potential problems in designing elastase inhibitors for 483

therapy. Am. Rev. Respir. Dis. 1991, 143, 1412-1415. 484

19. Prin-Mathieu, C.; Le Roux, Y.; Faure, G.; Laurent, F.; Béné, M.; Moussaoui, 485

F., Enzymatic activities of bovine peripheral blood leukocytes and milk 486

polymorphonuclear neutrophils during intramammary inflammation caused by 487

lipopolysaccharide. Clin. Diagn. Lab. Immunol. 2002, 9, 812-817. 488

20. Kelly, A.; O’Flaherty, F.; Fox, P., Indigenous proteolytic enzymes in milk: A 489

brief overview of the present state of knowledge. Int. Dairy J. 2006, 16, 563-572. 490

21. Hayes, M.; Stanton, C.; Fitzgerald, G. F.; Ross, R. P., Putting microbes to 491

work: Dairy fermentation, cell factories and bioactive peptides. Part II: Bioactive 492

peptide functions. Biotechnology journal 2007, 2, 435-449. 493

22. Wilkins, M. R.; Gasteiger, E.; Bairoch, A.; Sanchez, J.-C.; Williams, K. L.; 494

Appel, R. D.; Hochstrasser, D. F., Protein identification and analysis tools in the 495

ExPASy server. In 2-D Proteome Analysis Protocols, Springer: 1999; pp 531-552. 496

23. Christensen, B.; Schack, L.; Kläning, E.; Sørensen, E. S., Osteopontin is 497

cleaved at multiple sites close to its integrin-binding motifs in milk and is a novel 498

substrate for plasmin and cathepsin D. J. Biol. Chem. 2010, 285, 7929-7937. 499

500

501

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22

Figure Captions 502

503

Figure 1. Representation of the total number of cleaved milk proteins per predicted 504

enzyme. Each enzyme name on the X-axis is plotted against the total number of 505

proteins it cleaves on the Y-axis. 506

507

Figure 2. Illustration of antimicrobial-like peptides released in vivo from β-casein in 508

human milk. The peptide QELLLNPTHQIYPVTQPLAPVHNPISV shown at the top 509

of the figure in grey has been discovered to be an antimicrobial peptide (21). This 510

peptide is found at the C-terminus of β-casein (position 199 to 225). Four overlapping 511

peptides found to be released in vivo in human milk are represented under the β-512

casein sequence. The in vivo cleavage positions are indicated with grey lines. The 513

enzymes predicted to be responsible for these cleavages are represented beside the 514

grey lines. 515

516

517

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23

TABLES

Table 1. Details of the enzymes that take part in the digestion of the human milk proteins ranked based on their total number of cleavages. The

corresponding gene expression levels in RPKM are provided for each enzyme.

Enzymes

N-terminus

cleavage

count

C-terminus

cleavage

count

Total

cleavage

Unique

cleavage

Number of expected

cleavages within the

peptide

Number of

proteins cleaved

Odds

ratio

Std

error

Gene expression given in RPKM

Plasmin 120 200 320 0 371 15 4.11 1.08 0

Trypsin 1* 120 199 319 0 352 14 4.33 1.09

0

(5.81 PRKM for trypsin domain containing protease

TYSND1)

Cathepsin D 68 62 130 0 846 24 0.61 1.1 365.07

Chymotrypsin low

1

68 34 102 0 449 11 0.93 1.12 0

Elastase 51 39 90 0 563 13 0.64 1.12 0

Pepsin 1 (pH1.3) 31 47 78 0 479 10 0.66 1.13 0.1

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24

Glutamyl

endopeptidase*

55 21 76 0 930 5 0.31 1.13

0

(75.97 PRKM for proteasome subunit beta type-6,

responsible for the peptidyl glutamyl-like activity,

PSMB6)

Pepsin 1 (pH>2) 30 38 68 0 315 8 0.89 1.15 0.1

Proline

endopeptidase

14 31 45 0 598 18 0.29 1.17 5.52

Chymotrypsin low

4

3 12 15 0 102 4 0.60 1.32 0

Chymotrypsin low

3

6 6 12 0 79 6 0.62 1.36 0

Chymotrypsin low

2

2 0 2 0 0 1 20.65 4.71 0

Enzymes in bold represent ones that are not expressed at day 60 of lactation but low expression of these genes has been detected at earlier stages of lactation (15, and 30 days).

* This indicates enzymes that do not show expression in milk but expression data shows the presence of other enzymes that have a similar activity and are expressed.

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25

Table 2. Human milk proteins found to have been digested by plasmin. The proteins

are ordered based on the number of cleavages plasmin has performed on each. Here

we only considered the number of peptides that are unique, if a peptide was not

unique we consider only one copy of the peptide. Proteins exclusively found in milk

are represented in bold.

Protein name Uniprot ID and access name

Total number of cleavages

performed

β-casein P05814 (CASB_HUMAN) 89

Polymeric immunoglobulin

receptor

P01833 (PIGR_HUMAN) 32

Osteopontin P10451 (OSTP_HUMAN) 18

Butyrophilin subfamily 1 member

A1

Q13410 (BT1A1_HUMAN) 15

α-S1-casein P47710 (CASA1_HUMAN) 11

K-casein P07498 (CASK_HUMAN) 7

Mucin-1 P15941 (MUC1_HUMAN) 3

Parathyroid hormone-related

protein

P12272 (PTHR_HUMAN) 2

Bile salt-activated lipase P19835 (CEL_HUMAN) 2

Androgen receptor Q9UN21 (Q9UN21_HUMAN) 1

Protein diaphanous homolog 1 O60610 (DIAP1_HUMAN) 1

Complement C4-A P0C0L4 (CO4A_HUMAN) 1

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26

La-related protein 1 Q6PKG0 (LARP1_HUMAN) 1

NMDA receptor-regulated protein

2

Q659A1 (NARG2_HUMAN) 1

Dedicator of cytokinesis protein 1 Q14185 DOCK1_HUMAN 1

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27

Table 3. Human milk proteins found to have been digested by trypsin. The proteins

are ordered based on the number of cleavages trypsin has performed on each. Here we

only considered the number of peptides that are unique, if a peptide was not unique

we consider only one copy of the peptide. Proteins exclusively found in milk are

represented in bold.



performed


Polymeric immunoglobulin

receptor

P01833 (PIGR_HUMAN) 32


Butyrophilin subfamily 1

member A1

Q13410 (BT1A1_HUMAN) 15

αS1-casein P47710 (CASA1_HUMAN) 11

κκκκ-casein P07498 (CASK_HUMAN) 7

Mucin-1 P15941 (MUC1_HUMAN) 3

Parathyroid hormone-related

protein P12272 (PTHR_HUMAN) 2

Bile salt-activated lipase P19835 (CEL_HUMAN) 2



NMDA receptor-regulated Q659A1 (NARG2_HUMAN) 1

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28

protein 2

Complement C4-A P0C0L4 (CO4A_HUMAN) 1

Dedicator of cytokinesis protein

1

Q14185 (DOCK1_HUMAN) 1

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29

Table 4. Enzyme specificity for plasmin and trypsin.

Enzyme

Cleavage

Pattern

Reference

P4 P3 P2 P1 P1’ P2’

Trypsin - - W K P -

Wilkins et al.,

1999)

- - M R P - (22)

Plasmin - - - K or R - - (23)

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30

Table 5. Human milk proteins found to have been digested by cathepsin D. The

proteins are ordered based on the number of cleavages trypsin has performed on each.

Here we only considered the number of peptides that are unique, if a peptide was not

unique we consider only one copy of the peptide. Proteins exclusively found in milk

are represented in bold.

Protein name

Uniprot ID and access name

Total number

of cleavages

performed


Polymeric immunoglobulin receptor P01833 (PIGR_HUMAN) 21

Perilipin-2 Q99541 (PLIN2_HUMAN) 3



Deubiquitinating protein VCIP135 Q96JH7 (VCIP1_HUMAN) 2

Butyrophilin subfamily 1 member A1 Q13410 (BT1A1_HUMAN) 2

Receptor-type tyrosine-protein

phosphatase

Q15262 (PTPRK_HUMAN) 2

Macrophage mannose receptor 1 P22897 (MRC1_HUMAN) 2

Protein CASC3 O15234 (CASC3_HUMAN) 1

Flavin containing monooxygenase 5,

isoform CRA_c

Q9HA79 (Q9HA79_HUMAN) 1

Abl interactor 1 Q8IZP0 (ABI1_HUMAN) 1

Misshapen-like kinase 1 Q8N4C8 (MINK1_HUMAN) 1

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31



phosphatase α

P18433 (PTPRA_HUMAN) 1

Ubiquitin carboxyl-terminal hydrolase 51 Q70EK9 (UBP51_HUMAN) 1


Neural Wiskott-Aldrich syndrome protein O00401 (WASL_HUMAN) 1

κκκκ-casein P07498 (CASK_HUMAN) 1

Insulin receptor substrate 1 P35568 (IRS1_HUMAN) 1

Transcription factor 7-like 2 Q9NQB0 (TF7L2_HUMAN) 1

Gamma-glutamyltransferase 6 Q6P531 (GGT6_HUMAN) 1

PH domain leucine-rich repeat-containing

protein phosphatase 1

O60346 (PHLP1_HUMAN) 1

Dedicator of cytokinesis protein 1 Q14185 (DOCK1_HUMAN) 1

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32

Table 6. Human milk proteins found to have been digested by elastase. The proteins

are ordered based on the number of cleavages trypsin has performed on each. Here we

only considered the number of peptides that are unique, if a peptide was not unique

we consider only one copy of the peptide. Proteins exclusively found in milk are

represented in bold.



performed


Polymeric immunoglobulin receptor P01833 (PIGR_HUMAN) 25

Butyrophilin subfamily 1 member

A1

Q13410 (BT1A1_HUMAN) 8


Perilipin-2 Q99541 (PLIN2_HUMAN) 3

Protein CASC3 O15234 (CASC3_HUMAN) 1

Androgen receptor Q9UN21 (Q9UN21_HUMAN) 1

Parathyroid hormone-related protein P12272 (PTHR_HUMAN) 1


Heat shock protein β-1 P04792 (HSPB1_HUMAN) 1


phosphatase K

Q15262 (PTPRK_HUMAN) 1


phosphatase α P18433 (PTPRA_HUMAN) 1

Gamma-glutamyltransferase 6 Q6P531 (GGT6_HUMAN) 1

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FIGURES

Figure 1.

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http://pubs.acs.org/action/showImage?doi=10.1021/jf405601e&iName=master.img-000.jpg&w=390&h=269








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Figure 2.

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TOC Graphic

Graphic for Table of Contents

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predicting the important enzyme players in human breast ...€¦ · 26 whether these enzymes are...

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